Purinone derivative hydrochloride

ABSTRACT

The purinone derivative 6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochloride has Btk-selective inhibitory activity and, in addition to having excellent metabolic stability, it is a compound that exhibits a high level of solubility and absorption with respect to the free base and can be crystallized, hence it can serve as a therapeutic agent for diseases involving B cells and mast cells.

TECHNICAL FIELD

The present invention relates to6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride (hereinafter, referred to as the compound of the presentinvention), which has Btk inhibitory activity and is useful as atherapeutic agent for autoimmune diseases, cancer, and the like;crystals thereof; and a pharmaceutical composition containing the same.

BACKGROUND ART

Bruton's tyrosine kinase (abbreviated below as “Btk”) belongs to the Tecfamily of kinases, which are non-receptor tyrosine kinases, and isselectively expressed in the B cell and myelocyte lines. Btk plays animportant role in signal transduction in B cells and is a factor thatcontributes to the survival, differentiation, proliferation, andactivation of B cells. Signaling in B cells via the B cell antigenreceptor (BCR) induces a broad range of biological responses, andabnormal signal transduction here causes abnormal B cell activation andthe formation of pathogenic autoantibodies. Btk is believed to form alink in the BCR-mediated signal transduction pathways into B cells.Thus, X-linked agammaglobulinemia (XLA) is known to be caused by adefect in the human Btk gene that results in the induction of abnormal Bcell differentiation and a drastic decline in immunoglobulin production(refer to Non-patent Document 1). The symptoms of this disease include asubstantial decline in B cells in the peripheral blood and an increasedsusceptibility to bacterial infections. Btk is also known to participatein mast cell activation and in the physiological functions of platelets.Due to this, compounds that have a Btk inhibitory activity are effectivefor the treatment of diseases in which B cells or mast cellsparticipate, for example, allergic diseases, autoimmune diseases,inflammatory diseases, thromboembolic diseases, and cancers (refer toNon-patent Document 2).

The following compounds are known as prior art for the compounds of thepresent invention.

Compounds represented by general formula (A) are known as compounds thathave a Btk inhibitory activity

(in the formula, L_(a) ^(A) represents CH₂, O, NH, or S; Ar^(A)represents substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl; Y^(A) represents any substituent selected fromalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;Z^(A) represents CO, OCO, NHCO, or CS; R^(7-A) and R^(8-A) eachindependently represent H, unsubstituted C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstituted C₁-C₄ heteroalkyl, substituted C₁-C₄ heteroalkyl,unsubstituted C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,unsubstituted C₂-C₆ heterocycloalkyl, and substituted C₂-C₆heterocycloalkyl; or R^(7-A) and R^(8-A) together form a bond; andR^(6-A) represents H, substituted or unsubstituted C₁-C₄ alkyl,substituted or unsubstituted C₁-C₄ heteroalkyl, C₁-C₆ alkoxyalkyl, C₁-C₈alkylaminoalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, orsubstituted or unsubstituted aryl (the definitions of these groups havebeen excerpted)) (refer to Patent Documents 1, 2, and 3).

On the other hand, for example, compounds represented by general formula(B)

(in the formula, Q^(1B) and Q^(2B) are independently selected fromCX^(1B), CX^(2B), and nitrogen; Q^(3B) represents N or CH; X^(1B) andX^(2B) are independently selected from the group consisting of hydrogen,(C₁-C₆) alkyl, cyano, halogen, and so forth; R^(1B) is selected from thegroup consisting of hydrogen and (C₁-C₆) alkyl; yB represents 0 or aninteger from 1 to 3; R^(2B) and R^(3B) are independently selected fromhydrogen and (C₁-C₆) alkyl; R^(4B) is selected from the group consistingof alkyl, heterocyclyl, aryl, heteroaryl, and so forth; and R^(5B) isselected from the group consisting of alkyl, heterocyclyl, andsubstituted heterocyclyl (the definitions of these groups have beenexcerpted)) (refer to Patent Document 4) are known as compounds thathave a purinone skeleton.

Compounds represented by general formula (C) are also known

(in the formula, X^(C) is selected from the group consisting of nitrogenand CR^(8C); R^(8C) is selected from the group consisting of hydrogen,halogen, substituted or unsubstituted alkyl, and so forth; Q^(1c) isselected from the group consisting of O, S, and so forth; Z^(C) isselected from the group consisting of oxygen, sulfur, and NY^(5C);Y^(5C) is selected from the group consisting of hydrogen, substituted orunsubstituted alkyl, and so forth; Q^(2C), Q^(3C), and Q^(4C) areindependently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted aryl,and so forth; R^(2C) is selected from the group consisting of hydrogenand substituted or unsubstituted alkyl; and nC represents 0, 1, 2, 3, or4 (the definitions of these groups have been excerpted)) (refer toPatent Document 5).

In addition, Patent Document 6 discloses a compound having a purinonebackbone as Formula 20 (see paragraph [0028]).

The present invention relates to6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride, which has Btk-selective inhibitory activity, and inaddition to excellent metabolic stability, has greater solubility andabsorption than the free base; such matters are neither described norsuggested by the prior art documents.

-   Patent Document 1: Japanese Translation of PCT Application No.    2010-504324-   Patent Document 2: WO 2008/121742-   Patent Document 3: WO 2010/009342-   Patent Document 4: WO 2008/060301-   Patent Document 5: WO 2007/142755-   Patent Document 6: Japanese Translation of PCT Application No.    2003-509427-   Non-Patent Document 1: Nature, Vol. 361, pages 226-233, 1993-   Non-Patent Document 2: Anticancer Agents in Medicinal Chemistry,    Vol. 7, No. 6, pages 624-632, 2007

DISCLOSURE OF THE INVENTION

The problem to be solved by the present invention is the development ofa compound that has Btk-selective inhibitory activity, and in additionto excellent metabolic stability, has greater solubility and absorptionthan the free base in order to provide a very stable agent for thetreatment of a disease involving B cells and mast cells. In addition, acompound that has excellent stability as the active ingredient of apharmaceutical product and that can be crystalized to enable long-termstorage is preferred.

As the result of their diligent and incisive research to solve theaforementioned problem, the inventors discovered that the compound ofthe present invention has Btk-selective inhibitory activity, and inaddition to excellent metabolic stability, has greater solubility andabsorption than the free base, and can be crystallized, therebycompleting the present invention.

More specifically, the present invention relates to:

-   [1]    6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one    hydrochloride;-   [2] A crystal of    6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one    hydrochloride;-   [3] The crystal as in [2] above having at least 2 or more peaks at    angle 2θ selected from approximately 8.11, 8.43, 11.57, 12.73,    13.85, 14.20, 14.67, 14.91, 15.94, 16.64, 18.06, 19.74, 20.42,    21.05, 22.57, 23.21, 23.85, and 24.70 degrees in a powder x-ray    diffraction spectrum;-   [4] The crystal as in [2] or [3] above having peaks at angle 2θ of    approximately 8.11, 8.43, 14.20, 14.67, 14.91 and 23.21 degrees in a    powder x-ray diffraction spectrum;-   [5] The crystal as in any of [2] to [4] above having peaks at angle    2θ selected from approximately 8.11, 8.43, 11.57, 12.73, 13.85,    14.20, 14.67, 14.91, 15.94, 16.64, 18.06, 19.74, 20.42, 21.05,    22.57, 23.21, 23.85, and 24.70 degrees in a powder x-ray diffraction    spectrum;-   [6] The crystal as in any of [2] to [5] above which is characterized    by the powder x-ray diffraction spectral chart in FIG. 3;-   [7] The crystal as in any of [2] to [6] above having an endothermic    peak at a peak temperature of 216° C. in differential scanning    calorimetry;-   [8] The crystal as in any of [2] to [7] above which is characterized    by the differential scanning calorimetry chart in FIG. 4;-   [9] A pharmaceutical composition comprising    6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one    hydrochloride;-   [10] The pharmaceutical composition as in [9] above that is a Btk    inhibitor;-   [11] The pharmaceutical composition as in [10] above that is an    agent for the prevention and/or treatment of a Btk-related disease;-   [12] The pharmaceutical composition as in [11] above wherein the    Btk-related disease is an allergic disease, autoimmune disease,    inflammatory disease, thromboembolic disease, bone-related disease,    or cancer; and-   [13] The pharmaceutical composition as in [12] above wherein the    cancer is non-Hodgkin's lymphoma, etc.

The compound of the present invention has Btk-selective inhibitoryactivity, and in addition to having excellent metabolic stability, is acompound with greater solubility and absorption than the free base;therefore, it is useful as an outstandingly safe therapeutic agent for adisease involving B cells and mast cells such as non-Hodgkin's lymphoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a powder x-ray diffraction spectral chart of a crystal of6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one(in FIG. 1, the vertical axis represents intensity (counts), and thehorizontal axis represents 2θ (degrees));

FIG. 2 shows a differential scanning calorimetry (DSC) chart of acrystal of6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one;

FIG. 3 shows a powder x-ray diffraction spectral chart of a crystal of6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride (in FIG. 3, the vertical axis represents intensity(counts), and the horizontal axis represents 2θ (degrees)); and

FIG. 4 shows a differential scanning calorimetry (DSC) chart of acrystal of6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in greater detail below.

The term “Btk-selective inhibitory activity” means Btk-selectiveinhibitory activity with regard to tyrosine kinases other than Btk,particularly Lck, Fyn, and LynA. Due to this property, unexpectedadverse reactions caused by inhibiting other tyrosine kinases can beavoided.

In the present invention6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one(hereinafter, abbreviated as Compound A) means the compound representedby the following structural formula

(in the formula, the symbol

represents the β position).

[Study of Acid Addition Salts of Compound A]

Counter screening of acid addition salts of Compound A was performed bythe following method using Compound A prepared in Example 8 below andvarious acidic counter ions. Amorphous powders were obtained by mixing amolar amount of Compound A with equivalent amount of each acidic counterion; as step (I) methyl tert-butyl ether (MTBE) was added to the mixtureand the precipitate was scraped up with a micro-spatula, or as step (2),if no crystals precipitated in aforementioned step (1), methanol wasadded to the mixture, and it was allowed to dry naturally. When crystalswere obtained by this screening method, the physical property data wasmeasured under the conditions shown below. The physical property datafor Compound A was acquired beforehand, and that was compared with thephysical property data of the crystals obtained from the aforementionedcounter screening process.

[1] Powder x-Ray Diffraction Spectrum

<Measurement Conditions>

Device: BRUKER D8 DISCOVER with GADDS manufactured by Bruker AXS

Target: Cu Filter: None Voltage: 40 kV Current: 40 mA

Exposure time: 3 min

[2] Differential Scanning Calorimetry (DSC)

<Measurement Conditions>

Device: DSC 822e manufactured by Mettler ToledoSample weight: 1 to 2 mgSample cell: 40 μL aluminum panNitrogen gas flow rate: 40 mL/minRate of temperature increase: 10° C./min (25 to 240° C.)

The powder x-ray diffraction spectrum of the crystals of Compound A isshown in FIG. 1, and the differential scanning calorimetry (DSC) chartis shown in FIG. 2, respectively. Moreover, Table 1 below shows thediffraction angle 2θ and the relative intensity in the powder x-raydiffraction spectrum.

TABLE 1 Diffraction angle 2θ (degrees) Relative intensity (%) 5.88 10010.31 6.3 10.56 8.4 11.47 7.8 11.84 9.7 12.63 5.1 13.60 21.4 15.55 45.817.11 57 18.44 14.9 19.74 11.7 20.40 41.2 21.03 7.4 21.76 10.1 22.3914.3 22.77 10.1 23.48 3.5 24.00 4.5

Moreover, as shown in FIG. 2, the crystals of Compound A showedendothermic peaks corresponding to melting represented by an onsettemperature of approximately 169° C. and a peak temperature ofapproximately 172° C.

As shown in Table 2 below, the aforementioned screening was performedusing 18 species of acidic counter-ions.

TABLE 2 Acidic counter-ion Step (1) Step (2) Hydrochloric acid AmorphousOil Sulfuric acid Amorphous Oil Acetic acid Amorphous Crystals Citricacid Amorphous Crystals (+)-tartaric acid Oil Crystals Phosphoric acidAmorphous Crystals Fumaric acid Amorphous Crystals Lactic acid OilCrystals Succinic acid Oil Crystals Methanesulfonic acid Amorphous Oilp-toluenesulfonic acid Amorphous Oil Benzenesulfonic acid Amorphous Oil(−)-camphorsulfonic acid Amorphous Oil (+)-camphorsulfonic acidAmorphous Oil 2-naphthalenesulfonic acid Oil Oil 1-hydroxy-2-naphthoicacid Oil Crystals Benzoic acid Oil Crystals Nicotinic acid Nicotinicacid Crystals crystals

As a result, any crystals did not form a precipitate in step (1),meanwhile although crystals were obtained from 10 species of acidiccounter-ions (acetic acid, citric acid, (+)-tartaric acid, phosphoricacid, fumaric acid, lactic acid, succinic acid, 1-hydroxy-2-naphthoicacid, benzoic acid, and nicotinic acid) in step (2), these crystals allmatched the powder x-ray diffraction spectral chart of the crystal ofCompound A, so it was clear that no salts were formed thereby. On theother hand, a crystalline powder was not obtained from the remaining 8species of acidic counter-ions (hydrochloric acid, sulfuric acid,methanesulfonic acid, p-toluenesulfoinic acid, benzenesulfonic acid,(−)-camphorsulfonic acid, (+)-camphorsulfonic acid and2-naphthalenesulfonic acid), but because crystals of Compound A did notprecipitate, it appeared that salts had been formed. As a result, thefollowing crystallization study was carried out on these 8 species ofacidic counter-ions.

[Crystallization Study of Salts of Compound A]

A crystallization study of the salts of Compound A was carried out withan automatic crystallization device (Core Module X manufactured byFreeslate, Inc.) using sulfuric acid, methanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid as the acidiccounter-ions, and methanol, 2-propanol, acetone, toluene, ethyl acetate,acetonitrile, MTBE, and n-pentane as the solvents. For thecrystallization methods, four conditions were established, i.e. theslurry method (50° C., natural cooling to room temperature), thechilling method (50° C. to 10° C., decrease of −10° C./hour),precipitation method (dissolution at 50° C. followed by precipitation atroom temperature), and the evaporation enrichment method (dissolution at50° C. followed by evaporation at room temperature), and by combiningthe solvent and crystallization methods a total of 72 crystallizationconditions were established for each salt. As a result, however, acrystalline powder could not be obtained from any of the salts.

On the other hand, when a similar crystallization study was carried outusing hydrochloric acid as the acidic counter-ion, if 1,2-dimethoxyethane (DME) was used as the solvent a crystalline powder was obtained.When a powder x-ray diffraction measurement of the said crystallinepowder was performed under the aforementioned conditions, the peak shapewas different from that of the crystals of Compound A, and the resultsof ¹H-NMR and elemental analysis revealed these crystals were thehydrochloride of Compound A. Moreover, it was learned that because thepowder x-ray diffraction spectral patterns match, crystals of thehydrochloride of Compound A can be obtained even with the method ofExample 9 below. The powder x-ray diffraction spectrum of the crystalsof the compound prepared in Example 9 is shown in FIG. 3, and thedifferential scanning calorimetry (DSC) chart is shown in FIG. 4.Moreover, Table 3 below shows the diffraction angle 2θ and the relativeintensity in the powder x-ray diffraction spectrum. Among these, the 2θangles showed characteristic peaks at 8.11, 8.43, 14.20, 14.67, 14.91,and 23.21 degrees.

TABLE 3 Diffraction angle 2θ (degrees) Relative intensity (%) 8.11 21.88.43 20.6 11.57 52 12.73 19.1 13.85 30.4 14.20 45.3 14.67 33.4 14.9149.8 15.94 35.1 16.64 14 18.06 15 19.74 38.7 20.42 100 21.05 32.6 22.5732.4 23.21 46.7 23.85 48.6 24.70 29.3

Moreover, as shown in FIG. 4, the crystals of the compound prepared inExample 9 showed endothermic peaks corresponding to melting representedby an onset temperature of approximately 201° C. and a peak temperatureof approximately 216° C.

[Isomers]

The compound of the present invention can be either a 100% pure opticalisomer, or it can contain less than 50% of other optical isomers.

It is apparent to persons skilled in the art that, unless otherwisestated, in the present invention the symbol

represents bonding in the direction of the viewer of the page (i.e. theβ position), and

represents the α position, β position, or a mixture thereof in anyratio.

The compound of the present invention can be converted to a solvate. Thesolvate preferably has low toxicity and is water soluble. For example, asolvate of water or an alcohol system (e.g. ethanol, etc.) can be notedas a suitable solvate.

Moreover, the term “prodrug” of the compound of the present inventionrefers to a compound that is converted to the compound of the presentinvention in vivo by reacting with an enzyme, gastric acid, and thelike. For example, if the compound of the present invention has an aminogroup, a prodrug thereof can include a compound wherein the amino groupis acylated, alkylated, phosphorylated, and so on (e.g. a compoundwherein the amino group of the compound of the present invention iseicosanoylated, alanylated, pentylaminocarbonylated,(5-methyl-2-oxo-1,3-dioxolen-4-yl), methoxy carbonylated,tetrahydrofuranylated, pyrrolidinylmethylated, pivaloyloxymethylated,acetoxymethylated, tert-butylated, etc.) These compounds can be producedby publicly known methods. Moreover, a prodrug of the present inventioncan be either a hydrate or a non-hydrate. In addition, a prodrug of theinvention of the present invention can be one that converts into thecompound of the present invention under physiological conditions asdescribed in “Iyakuhin no Kaihatsu” Dai 7 kan, “Bunshi Sekkei,” pages163-198, Hirokawa Shoten 1990 [Drug Development” Vol. 7, “MolecularDesign,” pages 163-198, Hirokawa Shoten, 1990]. Furthermore, thecompound of the present invention can be labeled with an isotope (e.g.²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I,¹²⁵I, etc.)

[Toxicity]

The toxicity of the compound of the present invention is low enough thatit can be used safely as a pharmaceutical product.

[Application in a Pharmaceutical Product]

Because the compound of the present invention selectively inhibits Btk,it is useful as an agent for the prevention and/or treatment of diseasesthat involve Btk, i.e. diseases that involve B cells and mast cells, forexample, allergic diseases, autoimmune diseases, inflammatory diseases,thromboembolic diseases, bone-related diseases, cancer,graft-versus-host disease, and the like. Moreover, because the compoundof the present invention has the effect of selectively inhibiting B cellactivation, it is useful as a B cell activation inhibitor.

Examples of an allergic disease in the present invention includeallergy, anaphylaxis, allergic conjunctivitis, allergic rhinitis, atopicdermatitis and the like.

Examples of an autoimmune disease in the present invention includeinflammatory bowel disease, arthritis, lupus, rheumatoid arthritis,psoriatic arthritis, osteoarthritis, Still's disease, juvenilearthritis, type I diabetes, myasthenia gravis, Hashimoto's thyroiditis,Ord's thyroiditis, Basedow's disease, Sjogren's syndrome, multiplesclerosis, Guillain-Barre syndrome, acute disseminatedencephalomyelitis, Addison disease, opsoclonus-myoclonus syndrome,ankylosing spondylitis, antiphospholipid antibody syndrome, aplasticanemia, autoimmune hepatitis, celiac disease, Goodpasture's syndrome,idiopathic thrombocytopenic purpura, optic neuritis, scleroderma,primary biliary cirrhosis, Reiter's disease, Takayasu arteritis,temporal arteritis, warm autoimmune hemolytic anemia, Wegener granuloma,psoriasis, alopecia universalis, Burchett disease, chronic fatiguesyndrome, dysautonomia, endometriosis, interstitial cystitis, myotonia,vulvodynia, systemic lupus erythematosus, and the like.

Examples of an inflammatory disease in the present invention includeasthma, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis,cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis,cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis,endocarditis, endometritis, enteritis, epicondylitis, epididymitis,fasciitis, fibrositis, gastritis, gastroenteritis, hepatitis,hidradenitis suppurativa, laryngitis, mastitis, meningitis, myelitis,myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis,pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonia, proctitis, prostatitis, pyelonephritis,rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis,tonsillitis, uveitis, vaginitis, vasculitis, vulvitis, and the like.

Examples of a thromboembolic disease in the present invention includemyocardial infarction, angina pectoris, reocclusion after angioplasty,restenosis after angioplasty, reocclusion after aortocoronary bypass,restenosis after aortocoronary bypass, cerebral infarction, transientischemia, peripheral vascular occlusive disease, pulmonary embolism,deep vein thrombosis, and the like.

Examples of a bone-related disease in the present invention includeosteoporosis, periodontitis, metastasis of cancer to bone,osteoarthritis, hypercalcemia, bone fractures, Behcet's disease, and thelike.

Examples of cancer in the present invention include non-Hodgkin'slymphomas, and among those B cell non-Hodgkin's lymphoma is mostapplicable, for example, Burkitt's lymphoma, AIDS-related lymphoma,marginal zone B-cell lymphoma (nodal marginal zone B cell lymphoma,extranodal marginal zone B-cell lymphoma, splenic marginal zone B-celllymphoma), diffuse large B-cell lymphoma, primary effusion lymphoma,lymphoma-like granulomatous disease, follicular lymphoma, B-cell chroniclymphocytic leukemia, B cell prolymphocytic leukemia, lymphoplasmacyticleukemia/Waldenstrom's macroglobulinemia, plasmacytoma, mantle celllymphoma, mediastinal large B-cell lymphoma, intravascular large B-celllymphoma, and hairy cell leukemia. Moreover, examples of cancer in thepresent invention include cancers other than non-Hodgkin's lymphoma suchas pancreatic endocrine tumors and multiple myeloma. Examples ofpancreatic endocrine tumors include insulinoma, gastrinoma, glucagonoma,somatostatinoma, VIP-producing tumor (VIPoma), PP-producing tumor(PPoma), GRF-producing tumor, and the like.

The compound of the present invention can be administered together withanother drug as a concomitant medication to:

(1) supplement and/or enhance the preventive and/or therapeutic effectof the said compound;(2) improve the kinetics/absorption, or reduce the dose of the saidcompound; and/or(3) mitigate the side effects of the said compound.

The concomitant medication that contains the other drug, and thecompound of the present invention can be administered as a formulationthat combines both components therein or as a separate drug product.Administration thereof as a separate drug product includes bothadministration at the same time or administration at a different time.For administration at a different time, the compound of the presentinvention can be administered first followed by the other drug, or theother drug can be administered first followed by the compound of thepresent invention. The mode of administration of each can be the same ordifferent.

The disease for which the protective and/or therapeutic effect is to beprovided is not particularly limited herein as long as it is a diseasewherein the protective and/or therapeutic effect of the compound of thepresent invention is supplemented and/or enhanced by the aboveconcomitant medication.

Other drugs that supplement and/or enhance the protective and/ortherapeutic effect of the compound of the present invention againstallergic diseases include, for example, antihistamines, leukotrieneantagonists, anti-allergy drugs, thromboxane A2 receptor antagonist,thromboxane synthetase inhibitors, steroids and the like.

Other drugs that supplement and/or enhance the protective and/ortherapeutic efficacy of the compound of the present invention againstautoimmune diseases include, for example, immunosuppressants, steroids,disease-modifying antirheumatic drugs, elastase inhibitors,cannabinoid-2 receptor agonists, prostaglandins, prostaglandinsynthetase inhibitors, phosphodiesterase inhibitors, metalloproteinaseinhibitors, adhesion molecule inhibitors, anti-cytokine proteinpreparations such as anti-TNF-α preparations, anti-IL-1 preparations,and anti-IL-6 preparations, and cytokine inhibitors, non-steroidalanti-inflammatory drugs, anti-CD20 antibodies, and the like.

Other drugs that supplement and/or enhance the protective and/ortherapeutic efficacy of the compound of the present invention againstinflammatory diseases include, for example, steroids, elastaseinhibitors, cannabinoid-2 receptor agonists, prostaglandins,prostaglandin synthetase inhibitors, phosphodiesterase inhibitors,metalloproteinase inhibitors, adhesion molecule inhibitors,anti-leukotriene agents, anticholinergic agents, thromboxane A2 receptorantagonists, thromboxane synthase inhibitors, xanthine derivatives,expectorants, antibacterial agents, antihistamines, anti-cytokineprotein preparations, cytokine inhibitors, forskolin preparations,mediator release inhibitors, non-steroidal anti-inflammatory drugs, andthe like.

Other drugs that supplement and/or enhance the protective and/ortherapeutic efficacy of the compound of the present invention againstthromboembolic diseases include, for example, thrombolytic agents,heparin, heparinoids, low molecular weight heparin, warfarin, thrombininhibitors, factor Xa inhibitors, ADP receptor antagonists,cyclooxygenase inhibitors, and the like.

Other drugs that supplement and/or enhance the protective and/ortherapeutic efficacy of the compound of the present invention againstbone-related diseases include, for example, bisphosphonates,prostaglandins, vitamin D preparations, calcium preparations, estrogenpreparations, calcitonin preparations, ipriflavone preparations, proteinanabolic steroids, vitamin K preparations, cathepsin K inhibitors,parathyroid hormones, growth factors, caspase-1 inhibitors, PTHrPderivatives, metalloproteinase inhibitors, farnesoid X receptoragonists, anti-androgen agents, selective estrogen receptor modulators(SERMs), progesterone agonists, calcium receptor antagonists(calcylitics), strontium preparations, α-calcitonin gene-related peptidepreparations, osteogenetic protein preparations, anti-RANKL antibodies,anti-TNF-α antibodies, anti-IL-6 antibodies, and the like.

Other drugs that supplement and/or enhance the protective and/ortherapeutic efficacy of the compound of the present invention againstnon-Hodgkin's lymphoma include, for example, alkylating agents,antimetabolites, antitumor antibiotics, plant alkaloids, hormone drugs,platinum compounds, anti-CD20 antibody, other anti-cancer agents and thelike.

Examples of antihistamines include azelastine hydrochloride, ebastine,epinastine hydrochloride, emedastine fumarate, auranofin, oxatomide,olopatadine hydrochloride, dl-chlorpheniramine maleate, clemastinefumarate, ketotifen fumarate, cimetidine, dimenhyrinate, diphenhydraminehydrochloride, cyproheptadine hydrochloride, cetirizine hydrochloride,desloratadine, terfenadine, famotidine, fexofenadine hydrochloride,bepotastine, bepotastine besilate, mizolastine, mequitazine, mometasonefuroate, ranitidine, ranitidine hydrochloride, loratadine, promethazinehydrochloride, homochlorcyclizine hydrochloride, etc.

Examples of leukotriene antagonists include pranlukast hydrate,montelukast sodium, zafirlukast, ablukast, pobilukast, sulukast,iralukast sodium, verlukast, ritolukast, cinalukast, pirodomast,tomelukast, doqualast, and the like.

Examples of anti-allergy drugs include amlexanox, azelastinehydrochloride, israpafant, ibudilast, imitrodast sodium, ebastine,epinastine hydrochloride, emedastine fumarate, oxatomide, ozagrelhydrochloride, olopatadine hydrochloride, cromoglicic acid, sodiumcromoglicate, ketotifen fumarate, seratrodast, cetirizine hydrochloride,suplatast tosilate, tazanolast, terfenadine, domitroban calcium hydrate,tranilast, nedocromil, fexofenadine, fexofenadine hydrochloride,pemirolast potassium, mequitazine, ramatroban, repirinast, loratadine,and the like.

Examples of thromboxane A2 receptor antagonists include seratrodast,domitroban calcium hydrate, and ramatroban.

The thromboxane synthase inhibitors can be exemplified by imitrodastsodium and ozagrel hydrochloride.

Examples of steroids include amcinonide, hydrocortisone sodiumsuccinate, prednisolone sodium succinate, methylprednisolone sodiumsuccinate, ciclesonide, difluprednate, betamethasone propionate,dexamethasone, deflazacort, triamcinolone, triamcinolone acetonide,halcinonide, dexamethasone palmitate, hydrocortisone, flumetasonepivalate, prednisolone butylacetate, budesonide, prasterone sulfate,mometasonem furoate, fluocinonide, fluocinolone acetonide,fludroxycortide, flunisolide, prednisolone, alclometasone propionate,clobetasol propionate, dexamethasone propionate, deprodone propionate,fluticasone propionate, beclometasone propionate, betamethasone,methylprednisolone, methylprednisolone suleptanate, methylprednisolonesodium succinate, dexamethasone sodium phosphate, hydrocortisone sodiumphosphate, prednisolone sodium phosphate, diflucortolone valerate,dexamethasone valerate, betamethasone valerate, prednisolone valerateacetate, cortisone acetate, diflorasone acetate, dexamethasone acetate,triamcinolone acetate, paramethasone acetate, halopredone acetate,fludrocortisone acetate, prednisolone acetate, methylprednisoloneacetate, clobetasone butyrate, hydrocortisone butyrate, hydrocortisonebutyrate propionate, betamethasone butyrate propionate, and the like.

Examples of immunosuppressants include azathioprine, ascomycin,everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide,sirolimus, tacrolimus, bucillamine, methotrexate, leflunomide, and thelike.

Examples of disease-modifying anti-rheumatic drugs includeD-penicillamine, actarit, auranofin, salazosulfapyridine,hydroxychloroquine, bucillamine, methotrexate, leflunomide, lobenzaritsodium, aurothioglucose, sodium aurothiomalate, and the like.

Examples of elastase inhibitors include ONO-5046, ONO-6818, MR-889,PBI-1101, EPI-HNE-4, R-665, ZD-0892, ZD-8321, GW-311616, DMP-777,L-659286, L-680833, L-683845, AE-3763, and the like.

Examples of prostaglandins (hereinafter, abbreviated as PG) include PGE1drugs (e.g. alprostadil alfadex, alprostadil, etc.), PGI2 drugs (e.g.beraprost sodium, etc.), PG receptor agonists, PG receptor antagonists,and the like. Examples of PG receptors include PGE receptors (EP1, EP2,EP3, EP4), PGD receptors (DP, CRTH2), PGF receptors (FP), PGI2 receptors(IP), TX receptors (TP), and the like.

Examples of prostaglandin synthetase inhibitors includesalazosulfapyridine, mesalazine, olsalazine, 4-aminosalicylic acid,JTE-522, auranofin, carprofen, diphenpyramide, flunoxaprofen,flurbiprofen, indometacin, ketoprofen, lornoxicam, loxoprofen,meloxicam, oxaprozin, parsalmide, piproxen, piroxicam, piroxicamcinnamate, zaltoprofen, pranoprofen, and the like.

Examples of phosphodiesterase inhibitors include rolipram, cilomilast,Bay19-8004, NIK-616, roflumilast (BY-217), cipamfylline (BRL-61063),atizoram (CP-80633), ONO-6126, SCH-351591, YM-976, V-11294A, PD-168787,D-4396, IC-485 and the like.

Examples of adhesion molecule inhibitors include alpha4 integrinantagonists and the like.

Examples of anti-TNF-α preparations include anti-TNF-α antibodies,soluble TNF-α receptors, anti-TNF-α receptor antibodies, soluble TNF-αbinding proteins, and the like, and particularly infliximab andetanercept.

Examples of anti-IL-1 preparations include anti-IL-1 antibodies, solubleIL-1 receptors, anti-IL-1Ra antibodies and/or anti-IL-1 receptorantibodies, and the like, particularly anakinra.

Examples of anti-IL-6 preparations include anti-IL-6 antibodies, solubleIL-6 receptors, anti-IL-6 receptor antibodies, and the like,particularly tocilizumab.

Examples of cytokine inhibitors include suplatast tosylate, T-614,SR-31747, sonatimod, and the like.

Examples of anticholinergic agents include trihexyphenidyl,trihexyphenidyl hydrochloride, biperiden, biperiden hydrochloride, andthe like.

Examples of xanthine derivatives include aminophylline, theophylline,doxofylline, sipamphylline, diprophylline, and the like.

Examples of expectorants include foeniculated ammonia spirit, sodiumbicarbonate, bromhexine hydrochloride, carbocysteine, ambroxolhydrochloride, methylcysteine hydrochloride, acetylcysteine, L-cysteineethyl ester hydrochloride, tyloxapol, and the like.

Examples of antibacterials include sodium cefuroxime, meropenemtrihydrate, netilmicin sulfate, sisomicin sulfate, ceftibuten, PA-1806,IB-367, tobramycin, PA-1420, doxorubicin, astromicin sulfate, cefetametpivoxil hydrochloride, and the like.

Examples of mediator release agents include tranilast, sodiumcromoglicate, amlexanox, repirinast, ibudilast, dazanolast, pemirolastpotassium, and the like.

Examples of thrombolytic agents include alteplase, urokinase,tisokinase, nasaruplase, nateplase, t-PA, pamiteplase, monteplase,prourokinase, streptokinase, and the like.

An example of a heparinoid is fondaparinux.

Examples of low molecular weight heparins include danaparoid sodium,enoxaparin (sodium), nadroparin calcium, bemiparin (sodium), reviparin(sodium), tinzaparin (sodium), and the like.

Examples of thrombin inhibitors include argatroban, ximelagatran,melagatran, dabigatran, bivalirudin, lepirudin, hirudin, desirudin, andthe like.

Examples of ADP receptor antagonists include ticlopidine hydrochloride,clopidogrel sulfate, and the like.

Examples of cyclooxygenase inhibitors include aspirin and the like.

Examples of bisphosphonate preparations include alendronate sodiumhydrate, ibandronic acid, incadronate disodium, etidronate disodium,olpadronate, clodronate sodium hydrate, zoledronic acid, tiludronatedisodium, neridronate, pamidronate disodium, piridronate, minodronicacid hydrate, sodium risedronate hydrate, YM 175 and the like.

Examples of vitamin D preparations include alfacalcidol, falecalcitriol,calcitriol, 1α,25-dihydroxy cholecalciferol, dihydrotachysterol, ST-630,KDR, ED-71, rocaltrol, tacalciol, maxacalcitol and the like.

Examples of calcium preparations include calcium chloride, calciumgluconate, calcium glycerophosphate, calcium lactate, calciumL-aspartate, calcium hydrogen phosphate and the like.

Examples of estrogen preparations include estradiol, estradiol benzoate,estradiol cypionate, estradiol dipropionate, estradiol enanthate,estradiol hexahydrobenzoate, estradiol phenylpropionate, estradiolundecanoate, estradiol valerate, estrone, ethynyl estradiol, mestranoland the like.

Examples of calcitonin preparations include calcitonin, salmoncalcitonin, chicken calcitonin, secalciferol, elcatonin, TJN-135 and thelike.

Examples of ipriflavone preparations include ipriflavone and the like.

Examples of protein anabolic steroids include oxymetholone, stanozolol,nandrolone decanoate, nandrolone phenylpropionate, nandrolonecyclohexylpropionate, metenolone acetate, mestanolone, ethylestrenol,calusterone and the like.

Examples of vitamin K preparations include menatetrenone, phytonadioneand the like.

Examples of cathepsin K inhibitors include ONO-5334, AAE 581, SB 462795,and odanacatib, and the like.

Examples of parathyroid hormone (PTH) include dried thyroid,levothyroxine sodium, liothyronine sodium, propylthiouracil, thiamazole,teriparatide acetate and the like.

Examples of growth factors include fibroblast growth factor (FGF),vascular endothelial growth factor (VEGF), hepatocyte growth factor(HGF), insulin-like growth factor (IGF) and the like.

Examples of caspase-1 inhibitors include nitroflubiprofen, pralnacasanand the like.

Examples of PTHrP derivatives include hPTHrP, RS-66271 and the like.

Examples of the farnesoid X receptor agonists include SR-45023A and thelike.

Examples of anti-androgen agents include osateron acetate and the like.

Examples of selective estrogen receptor modulators (SERMs) includeTSE-424, WJ-713/MPA, lasofoxifene tartrate, raloxifene hydrochloride,tamoxifen citrate and the like.

Examples of progesterone agonist include trimegestone and the like.

Examples of calcium receptor antagonists (calcilytics) includeNPS-423557 and the like.

Examples of strontium preparations include strontium ranelate and thelike.

Examples of anti-RANKL antibodies include denosumab (AMG 162) and thelike.

Examples of osteogenetic protein preparations include YM 484 and thelike.

Examples of alkylating agents include nitrogen mustard N-oxidehydrochloride, cyclophosphamide, Ifosfamide, melphalan, thiotepa,carboquone, busulfan, nimustine hydrochloride, dacarbazine, ranimustine,and the like.

Examples of antimetabolites include methotrexate, mercaptopurine,6-mercaptopurine riboside, fluorouracil, tegafur, tegafur uracil,carmofur, doxifluridine, cytarabine, enocitabine, tegafur gimestatotastat potassium, gemcitabine hydrochloride, cytarabine ocfosfate,procarbazine hydrochloride, hydroxycarbamide, and the like.

Examples of anticancer antibiotics include actinomycin D, mitomycin C,daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicinhydrochloride, neocarzinostatin, pirarubicin hydrochloride, epirubicin(hydrochloride), idarubicin hydrochloride, chromomycin A3, bleomycin(hydrochloride), peplomycin sulfate, therarubicin, zinostatinstimalamer, and the like.

Examples of plant preparations include vinblastine sulfate, vincristinesulfate, vindesine sulfate, irinotecan hydrochloride, etoposide,flutamide, vinorelbine tartrate, docetaxel hydrate, paclitaxel, and thelike.

Examples of hormones include estramustine phosphate sodium,mepitiostane, epitiostanol, goserelin acetate, fosfestrol(diethylstilbestrol phosphate), tamoxifen citrate, toremifene citrate,fadrozole hydrochloride hydrate, medroxyprogesterone acetate,bicalutamide, leuprorelin acetate, anastrozole, exemestane, and thelike.

Examples of platinum compounds include carboplatin, cisplatin,nedaplatin, and the like.

Examples of anti-CD20 antibodies include rituximab, ibritumomab,ocrelizumab, and the like.

Examples of other anticancer agents include L-asparaginase, octreotideacetate, porfimer sodium, mitoxantrone acetate, and the like.

The concomitant medication used together with the compound of thepresent invention can include not only drugs that have been discoveredto date, but also drugs that may be discovered in the future.

The compound of the present invention is generally administeredsystemically or locally, and as an oral or parenteral form. Examples oforal formulations include liquids for oral administration (e.g. elixirs,syrups, pharmaceutically acceptable water-based formulations,suspensions, and emulsions) and solids for oral administration (e.g.tablets (including sublingual tablets and orally disintegratingtablets), pills, capsules (including hard capsules, soft capsules,gelatin capsules, and microcapsules), powders, granules, and lozenges),and the like. Examples of parenteral formulations include solutions(e.g. injectables (such as subcutaneous injectables, intravenousinjectables, intramuscular injectables, intraperitoneal injectables, anddrip formulations), eye drops (e.g. aqueous eye drops (such as aqueouseye drops, aqueous eye drop suspensions, viscous eye drops, solubilizedeye drops, etc.) and nonaqueous eye drops (such as nonaqueous eye dropsand nonaqueous eye drop suspensions, etc.)), topical formulations (e.g.ointments (such as ophthalmic ointments, etc.)), ear drops), and thelike. These preparations can be controlled release formulations such asrapid release formulations, sustained release formulations, and thelike. These preparations can be produced by publicly known methods suchas the methods described in The Japanese Pharmacopoeia.

As agents for oral administration, the liquid preparations for oraladministration can be produced, for example, by dissolving, suspending,or emulsifying the compound of the present invention in a commonly useddiluent (e.g. purified water, ethanol, or a mixture thereof, etc.).These liquid preparations may also contain a wetting agent, suspendingagent, emulsifier, sweetener, flavoring, fragrance, preservative,buffer, and the like.

As a solid for oral administration, the solid oral preparations can beprepared by mixing the compound of the present invention with, anexcipient (e.g. lactose, mannitol, glucose, microcrystalline cellulose,starch, etc.), a binder (e.g. hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.), a disintegrant(e.g. cellulose calcium glycolate, etc.), a lubricant (e.g. magnesiumstearate, etc.), a stabilizer, a solubilizer (e.g. glutamic acid,aspartic acid, etc.), and the like, and formulating according toconventional methods. As needed, coating can be carried out with acoating agent (e.g. sugar, gelatin, hydroxypropyl cellulose,hydroxypropyl methylcellulose phthalate, etc.) and two or more layerscan be applied.

As parenteral preparations, topical preparations can be produced using apublicly known method and a commonly used formulation. For example, anointment can be prepared by incorporating or dissolving the compound ofthe present invention into a base. The ointment base can be selectedfrom publicly known ointment bases or a commonly used ointment base. Forexample, one item alone or a mixture of two or more items selected fromthe following can be used: higher fatty acids and higher fatty acidesters (e.g. adipic acid, myristic acid, palmitic acid, stearic acid,oleic acid, adipate esters, myristate esters, palmitate esters, stearateesters, oleate esters, etc.), waxes (e.g. beeswax, spermaceti, ceresin,etc.), surfactants (e.g. polyoxyethylene alkyl ether phosphate esters,etc.), higher alcohols (e.g. cetanol, stearyl alcohol, cetostearylalcohol, etc.), silicone oils (e.g. dimethyl polysiloxane, etc.),hydrocarbons (e.g. hydrophilic petrolatum, white petrolatum, purifiedlanolin, liquid paraffin, etc.), glycols (e.g. ethylene glycol,diethylene glycol, propylene glycol, polyethylene glycol, macrogol,etc.), plant oils (e.g. castor oil, olive oil, sesame oil, turpentineoil, etc.), animal oils (e.g, mink oil, egg yolk oil, squalane,squalene, etc.), water, absorption promoters, and anti-irritants. Ahumectant, preservative, stabilizer, antioxidant, fragrance, and thelike may also be included therein.

As parenteral preparations, injectables include solutions, suspensions,and emulsions as well as injectables in solid form to be used afterdissolution or suspension in a solvent at the time of use. For example,an injectable can be used by dissolving, suspending, or emulsifying thecompound of the present invention in a solvent. Examples of the solventinclude distilled water for injection, physiological saline solution,vegetable oil, propylene glycol, polyethylene glycol, an alcohol such asethanol, or a combination thereof. The injectable can also contain astabilizer, a solubilizer (e.g. glutamic acid, aspartic acid,Polysorbate 80®, etc.), a suspending agent, an emulsifier, a soothingagent, a buffer, a preservative, and the like. The injectable can besterilized in the final process or can be manufactured using asepticprocessing methods. The injectable can also be manufactured as a sterilesolid form, for example, a freeze-dried product, and can be used afterdissolution in distilled water for injection or another solvent that iseither sterile or sterilized prior to use.

The dose of the compound of the present invention can be selectedappropriately depending on the condition, age, type of formulation, andthe like, and in the case of an oral preparation preferably 1 to 100 mg,or more preferably 5 to 30 mg can be administered 1 to several times aday (e.g. 1 to 3 times). Moreover, the compound of the present inventioncan be administered parenterally 1 to several times a day in a range of50 μg to 500 mg per dose, or can be continuously administeredintravenously in a range from 1 to 24 hours per day.

Of course, as noted above, the dose will depend upon various conditionsand, as a result, cases will occur wherein an amount less than the abovedosage will be sufficient or cases will occur wherein those ranges mustbe exceeded.

EXAMPLES

The present invention is described in detail below through examples, butis by no means limited thereto.

The solvents in parentheses shown in the sections on chromatographicseparation and TLC indicate the elution solvent or development solventthat was used, and the ratio represents the ratio by volume.

Unless otherwise stated, the NMR data is ¹H-NMR data.

The items in parentheses shown in the NMR sections represent thesolvents used in measurement.

The compound names used in this Description are generally namesgenerated based on IUPAC nomenclature or generated using ACD/Name®, acomputer program from Advanced Chemistry Development, Inc., thatperforms naming based on IUPAC rules.

Example 1 N,N-dibenzyl-6-chloro-5-nitropyrimidine-4-amine

A solution of dibenzylamine (10.2 g) in dichloromethane (30 mL) wasdripped into a solution of 4,6-dichloro-5-nitropyrimidine (10 g) indichloromethane (70 mL) on an ice bath. Then triethylamine (14.4 mL) wasadded, and the mixture was stirred for 1 hour. Water was added to thereaction mixture, the organic layer was washed with a saturated aqueoussodium chloride solution and dried over anhydrous sodium sulfate, andthe solvent was concentrated under reduced pressure to obtain the titlecompound (19.2 g) with the physical property value shown below.

TLC: Rf 0.50 (hexane:ethyl acetate=7:1).

Example 2 tert-butyl(3R)-3-{[6-(dibenzylamino)-5-nitropyrimidin-4-yl]amino}pyrrolidine-1-carboxylate

The compound prepared in Example 1 (19 g) and tert-butyl(3R)-3-aminopyrrolidine-1-carboxylate (10.5 g) were dissolved in dioxane(58 mL). Triethylamine (8.1 mL) was added, and the mixture was stirredfor 5 hours at 50° C. The reaction mixture was returned to roomtemperature, the solvent was distilled off, water was added, andextraction was performed with ethyl acetate. The organic layer waswashed with saturated aqueous sodium chloride solution, then dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified by silica gel column chromatography to obtain the titlecompound (27.0 g) with the physical property value shown below.

TLC: Rf 0.29 (hexane:ethyl acetate=4:1)

Example 3 tert-butyl(3R)-3-{[5-amino-6-(dibenzylamino)pyrimidin-4-yl]amino}pyrrolidine-1-carboxylate

An ethyl acetate (360 mL) solution of the compound prepared in Example 2(17.5 g) was dripped into a mixture of zinc (23.3 g) and a 3.0 M aqueousammonium chloride solution (11.4 g) on an ice bath, and the temperaturewas immediately raised to room temperature. After stirring for 2 hours,the reaction mixture was filtered through Celite™ and the solvent wasdistilled off. The residue was purified by silica gel columnchromatography to obtain the title compound (12.4 g) with the physicalproperty value shown below.

TLC: Rf 0.69 (hexane:ethyl acetate=1:1)

Example 4 tert-butyl(3R)-3-[6-(dibenzylamino)-8-oxo-7,8-dihydro-9H-purin-9-yl]pyrrolidin-1-carboxylate

The compound prepared in Example 3 (8.4 g) and 1,1′-carbonyl diimidazole(5.9 g) were dissolved in tetrahydrofuran (120 mL) and the solution wasstirred for 15 hours at 60° C. The solvent was distilled off from thereaction mixture, water was added, and extraction with ethyl acetate wasperformed. The organic layer was washed with saturated aqueous sodiumchloride solution, then dried over anhydrous sodium sulfate, and thesolvent was distilled off. The residue was purified by silica gel columnchromatography to obtain the title compound (7.8 g) with the physicalproperty value shown below.

TLC: Rf 0.28 (hexane:ethyl acetate=2:1)

Example 5 tert-butyl(3R)-3-(6-amino-8-oxo-7,8-dihydro-9H-purin-9-yl)pyrrolidine-1-carboxylate

The compound prepared in Example 4 (7.8 g) was dissolved in methanol(240 mL) and ethyl acetate (50 mL), 20% Pearlman's catalyst (Pd(OH)₂/C)(8.0 g, 100 wt %) was added, hydrogen gas replacement was carried out,and stirring was performed for 7.5 hours at 60° C. The reaction mixturewas filtered through Celite™ and the solvent was distilled off to obtainthe title compound (5.0 g) with the physical property value indicatedbelow.

TLC: Rf 0.50 (ethyl acetate)

Example 6 tert-butyl(3R)-3-[6-amino-8-oxo-7-(4-phenoxyphenyl)-7,8-dihydro-9H-purin-9-yl]pyrrolidine-1-carboxylate

At room temperature p-phenoxy phenyl boronic acid (2.1 g), copper(II)acetate (1.48 g), molecular sieve 4A (2.5 g), and pyridine (0.82 mL)were added to a dichloromethane suspension (200 mL) of the compoundprepared in Example 5 (2.5 g), followed by stirring for 21 hours. Thereaction mixture was filtered through Celite™ and the residue waspurified by silica gel column chromatography to obtain the titlecompound (1.3 g) with the physical property value shown below.

TLC: Rf 0.18 (hexane:ethyl acetate=1:1)

Example 7(3R)-6-amino-9-pyrrolidin-3-yl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onedihydrochloride

At room temperature 4 N HCl/dioxane (13 mL) was added to a methanol (13mL) suspension of the compound prepared in Example 6 (1.3 g 2.76 mmol,1.0 equivalent), and the mixture was stirred for 1 hour. The solvent wasthen distilled off to obtain the title compound (1.5 g) with thephysical property value shown below.

TLC: Rf 0.50 (dichloromethane: methanol:28% ammonia water=9:1:0.1)

Example 86-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one(Compound A)

After 2-butylnoic acid (34 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (78 mg), 1-hydroxybenzotriazole (HOBt)(62 mg), and triethylamine (114 μL) were added to a solution of thecompound prepared in Example 7 (100 mg) in dimethyl formamide (3 mL),the mixture was stirred at room temperature for 3 hours. Water was addedto the reaction mixture and extraction with ethyl acetate was performed.The organic layer was washed with saturated sodium carbonate solutionand saturated aqueous sodium chloride solution, then dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified by thin layer chromatography (dichloromethane:methanol:28%ammonia water=90:10:1) to obtain the title compound (75 mg) with thephysical property values shown below.

TLC: Rf 0.68 (ethyl acetate:methanol=9:1);

1H-NMR (CDCl₃):δ 1.94-2.03, 2.23-2.39, 2.80-3.01, 3.50-3.63, 3.67-3.80,3.86-4.02, 4.03-4.18, 4.23-4.33, 4.42-4.51, 5.11-5.25, 7.04-7.23,7.34-7.45, 8.20-8.23

Example 96-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride

The compound prepared in Example 8 (3.0 g) was placed in a 300 mL 3-neckpear-shaped flask, ethyl acetate (30 mL) and 1-propanol (4.5 mL) wereadded, and the external temperature was set at 70° C. (internaltemperature 61° C.). After it was confirmed that the compound preparedin Example 8 had dissolved completely, 10% HCl/methanol (3.5 mL) wasadded, and after precipitation of crystals was confirmed, the crystalswere ripened by the following sequence: external temperature 70° C. for30 min, external temperature 60° C. for 30 min, external temperature 50C for 60 min, external temperature 40 C for 30 min, room temperature for30 min, and on an ice bath for 30 min. The resulting crystals werefiltered, washed with ethyl acetate (6 mL), and dried under vacuum at50° C. to obtain white crystals of the title compound (2.76 g) with thephysical property values shown below.

TLC: Rf 0.55 (dichloromethane:methanol=9:1);

1H-NMR (CD₃OD):δ 1.97-2.07, 2.38-2.52, 2.63-2.80, 3.51-3.63, 3.77-3.94,4.00-4.19, 4.27-4.35, 5.26-5.38, 7.08-7.23, 7.38-7.52, 8.44-8.47

Pharmacological Test Examples Biological Example 1 Measurement of BtkInhibitory Activity and Selectivity Toward Btk (In Vitro)

The measurement of Btk enzyme inhibitory activity was performed usingthe following reagents: Z′-LYTE™ Kinase Assay Kit-Tyr 1 (containing Tyr1 peptide, Thy 1 phospho-peptide, 5× kinase buffer, ATP, developmentreagent B, development buffer, and stop reagent), Tyr 1 peptide(Invitrogen™), and Btk (Invitrogen™) according to the instructionsaccompanying the kit.

First 5 μL/well of either a solution prepared by diluting the testcompound in dimethyl sulfoxide (DMSO) or DMSO alone was added to thewells of a 96-well assay plate together with 10 μL/well ofsubstrate/enzyme mixture and allowed to react for 20 minutes at 30° C.The substrate/enzyme mixture solutions were prepared by dilution inkinase buffer (DL-dithiothreitol (DTT; 2.7 mM) and 1.33× kinase buffer)so that the final concentration of Tyr-1 peptide would be 4 μM, and thefinal concentration of Btk would be 5 nM. Next 5 μL/well of adenosinetriphosphate was added (ATP; final concentration 36 μM) and reacted for1 hour at 30° C. After the reaction was completed, 10 μL of developmentsolution prepared by diluting development reagent B in developmentbuffer 128-fold was added and reacted for an additional 1 hour at 30° C.Then the enzyme reaction was stopped by adding 10 μL of stop solution.The fluorescence intensity in each well was measured using afluorescence plate reader (Fusion Universal Microplate Analyzer,PerkinElmer, Inc.) at 520 nm and 445 nm. In accordance with theinstructions accompanying the kit, the phosphorylation rate wasdetermined by the ratio of the emission at 445 nm (coumalin emission) inrelation to emission at 520 nm (fluorescein emission).

The inhibition (%) was calculated from the following formula.

Phosphorylation inhibition rate (%)=1−{(A _(C) −A _(X))/(A _(C) −A_(B))}×100  [Mathematical Formula 1]

A_(X): Phosphorylation rate when test compound was addedA_(B): Phosphorylation rate without ATP (blank)A_(C): Phosphorylation rate with DMSO alone (control)

The 50% inhibition rate of the test compound (IC₅₀ value) was calculatedfrom an inhibition curve based on the rate of inhibition at eachconcentration of the test compound.

Measurements of the inhibitory activity on other kinases were made inthe same manner as described above using various kinases such as Lck,Fyn, LynA (Invitrogen Corporation) in place of Btk.

Results revealed that the IC₅₀ value of the compound prepared in Example9 was 0.0021 μM.

In addition, Btk-selective inhibitory activity rates of the compoundprepared in Example 9 toward other kinases, particularly Lck, Fyn, andLynA, were calculated on the basis of the IC₅₀ values for each kinase,and these are shown in Table 4 below.

TABLE 4 Lck[IC₅₀]/ Btk[IC₅₀] Fyn[IC₅₀]/Btk[IC₅₀] LynA[IC₅₀]/Btk[IC₅₀]Example 9 375 1057 1662

Results show that the compound of the present invention not only has Btkinhibitory activity, but also has Btk-selective inhibitory activitytoward other kinases.

Biological Example 2 Blood Kinetics in Dogs

The kinetic profiles of compound A and the salt thereof (the compoundprepared in Example 9) in blood were evaluated in male beagle dogs underfasting conditions. Intravenous administration, as well as both oralliquid and oral suspension administrations were carried out for CompoundA prepared in Example 8, and oral administration by capsule was carriedout for the compound prepared in Example 9. For the intravenous and oralsolution administrations, a solubilized solution of Compound A dissolvedin WellSolve (Celeste Inc.) heated to 60° C. was used. Using thesolubilized solutions, doses of 1 mg/1 mL/kg were rapidly administeredintravenously with a syringe via a forelimb cephalic vein, and doses of1 mg/5 mL/kg were administered by gavage using a catheter. Approximately300 μL blood samples were drawn from the jugular vein as follows: forintravenous administration-before administration, 2, 5, 15, and 30minutes after administration, and 1, 2, 4, 6, 8, and 24 hours afteradministration; for oral administration-before administration, 5, 15,and 30 minutes after administration, and 1, 2, 4, 6, 8, and 24 hoursafter administration. The blood samples were cooled on ice, separated bycentrifuge at 12,000 rpm for 3 min, and the plasma was collected. Theconcentration of Compound A in the plasma was measured by LC/MS/MS(UPLC/Xevo, Waters). The area under the curve (AUC, ng·h/mL), maximumconcentration (C_(max), μg/mL) and clearance (CL, mL/hr/kg) werecalculated from the plasma concentration. The bioavailability (BA) ofCompound A was calculated from the AUC of the oral dose and the AUC ofthe intravenous dose. It was found that the BA of Compound A using thesolubilized solution was 114.6%.

Moreover, for the oral suspension administration a liquid was preparedby finely pulverizing Compound A and suspending the particles in anaqueous solution of 0.5% methylcellulose, and then administering dosesof 3 and 10 mg/kg in the same manner as the forced oral administrationby gavage described above. The compound from Example 9 was mixed at a1:1 ratio with D-mannitol to make a dose of 3 mg/kg and placed in a No.4 capsule (Qualicaps Co., Ltd.). The capsule was administered orally byplacing 3 mg/capsule doses deep in the throat to avoid chewing, holdingthe mouth closed, and inserting 50 mL of water for injection between theteeth to force swallowing. The timing of plasma sampling and measurementof the plasma concentration of Compound A for each administration methodwere performed in the same manner as in the oral administration ofCompound A using the solubilized solution that was described above. TheBA values for each administration method and at each concentration werecalculated as relative BA by assigning the BA of Compound A in thesolubilized solution a value of 100%. The results are shown in Table 5below.

TABLE 5 Dose Relative (mg/kg) BA (%) Solubilized solution of Compound A1 100 Suspension of pulverized Compound A 3 42 Suspension of pulverizedCompound A 10 27 Capsule of compound prepared in Example 9 3 86

The results show that the relative BA of the liquid suspension ofpulverized Compound A decreased as the dose increased. Conversely, therelative BA of the compound prepared in Example 9 was higher than theliquid suspension of pulverized Compound A at the same dose. Therefore,it was found that the absorption of the compound of the presentinvention surpasses that of Compound A.

Biological Example 3 Measurement of Solubility

First 0.5 mg to 2.5 mg of Compound A (pulverized by a Jet Mill) and thecompound prepared in Example 9 were placed in 2.5 mL of various solvents(Japanese Pharmacopoeia dissolution test I liquid, JapanesePharmacopoeia dissolution test II liquid, diluted McIlvaine buffer (pH4.0, pH 7.4), purified water, and artificial intestinal fluids (FaSSIF,FeSSIF). With stirring at 700 rpm with a magnetic stirrer, approximately1 mL samples were taken from the test suspension at 30 min and 24 hafter the start of the test, and after filtering with a 0.2 μm filter,the solubility of Compound A was measured by HPLC under the conditionsshown below. Table 6 below compares the solubility of Compound A and thecompound prepared in Example 9.

<HPLC Measurement Conditions>

Device: HPLC 1100series, AgilentColumn: YMC-Pack ODS-AM AM-302 (4.6 mm ID×150 mm long)Column temperature: 25° C.Mobile phase: 20 mM potassium phosphate monobasic (pH 3.0)/acetonitrile(60:40, isocratic)

UV: 210 nm

Flow rate: 1.0 mL/minSample rack temperature: 25° C.Sample injection volume: 10 μLMeasurement time: 12 minRetention time: 8.9 min

TABLE 6 Solubility (μg/mL) Compound A Example 9 After After After After30 min 24 h 30 min 24 h JP dissolution test I liquid 550 717 1223 1208(pH 1.2) Dilute McIlvaine buffer 30 34 125 117 (pH 4.0) JP dissolutiontest II liquid 31 32 112 111 (pH 6.8) Dilute McIlvaine buffer 29 29 103106 (pH 7.4) Purified water 32 34 130 144 FaSSIF (pH 6.5) 35 36 145 118FeSSIF (pH 6.5) 67 84 356 301

From the above it is clear that the compound of the present inventionhas greater solubility in all of the solvents than Compound A.

Biological Example 4 Evaluation of Stability in Rat and Human LiverMicrosomes (1) Preparation of Test Compound Solution

A 0.25 mmol/L solution was prepared by diluting the test compound (5 μLof 10 mmol/L DMSO solution) in 50% acetonitrile/water solution (195 μL).

(2) Preparation of 0 Min-Reaction Sample

First 245 μL of 0.1 mol/L phosphate buffer (pH 7.4) containing 0.5 mg/mLof rat and human liver microsomes (Xenotech) and NADPH-Co-factor (BDBiosciences) was placed in a reaction vessel that had been pre-heated to37° C. and pre-incubated for 5 min. Then the test compound solution (5μL) was added to start the reaction. Immediately after the reaction wasstarted, a 20 μL sample was taken, 180 μL of acetonitrile containing theinternal reference substance (warfarin) was added, and the reaction wasstopped. After the 20 μL of solution was stirred together with 180 μL of50% acetonitrile/water solution on a plate equipped with a proteinextraction filter, the liquid was suction-filtered and used as thestandard sample.

(3) Preparation of 15 Min-Reaction Sample

After it was incubated for 15 min at 37° C., 20 μL of the above reactionsolution was added to 180 μL of cold acetonitrile (containing internalreference substance warfarin), and the reaction was stopped. After the20 μL of solution was stirred together with 180 μL of 50%acetonitrile/water solution on a plate equipped with a proteinextraction filter, the liquid was vacuum-filtered and used as thestandard sample.

(4) Evaluation Method and Results

First 1 μL of sample solution was injected into the LC-MS/MS, and theresidual amount (%) was calculated by dividing the ratio of the peakarea of the reaction sample (peak area of test compound/peak area ofinternal reference substance) by the peak area ratio of the standardsample and multiplying by 100.

Formulation Examples Formulation Example 1

The components shown below were mixed by a conventional method andtableted to obtain 10,000 tablets containing 10 mg of active ingredientper tablet.

6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4- 100 gphenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochlorideCarboxymethylcellulose potassium (disintegrant)  20 g Magnesium stearate(lubricant)  10 g Microcrystalline cellulose 870 g

Formulation Example 2

The components shown below were mixed by a conventional method, filteredthrough a dust filter, filled into 5 mL ampoules, and heat sterilizedwith an autoclave to obtain 10,000 ampoules containing 20 mg of activeingredient per ampoule.

6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4- 200 gphenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochloride Mannitol  20 gDistilled water  50 L

INDUSTRIAL APPLICABILITY

In addition to having Btk-selective inhibitory activity, the compound ofthe present invention has excellent metabolic stability, is a compoundwith greater solubility and absorption than the free base, and can becrystallized; therefore, it is useful as a therapeutic agent for adisease involving B cells and mast cells such as non-Hodgkin's lymphoma.

1.6-Amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride.
 2. A crystal of6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride.
 3. The crystal according to claim 2, having at least 2 ormore peaks at angle 20 selected from approximately 8.11, 8.43, 11.57,12.73, 13.85, 14.20, 14.67, 14.91, 15.94, 16.64, 18.06, 19.74, 20.42,21.05, 22.57, 23.21, 23.85, and 24.70 degrees in a powder x-raydiffraction spectrum.
 4. The crystal according to claim 2, having peaksat angle 2θ of approximately 8.11, 8.43, 14.20, 14.67, 14.91 and 23.21degrees in a powder x-ray diffraction spectrum.
 5. The crystal accordingto claim 2, having peaks at angle 2θ selected from approximately 8.11,8.43, 11.57, 12.73, 13.85, 14.20, 14.67, 14.91, 15.94, 16.64, 18.06,19.74, 20.42, 21.05, 22.57, 23.21, 23.85, and 24.70 degrees in a powderx-ray diffraction spectrum.
 6. The crystal according to claim 2, whichis characterized by the powder x-ray diffraction spectral chart in FIG.3.
 7. The crystal according to claim 2, having an endothermic peak at apeak temperature of 216° C. in differential scanning calorimetry.
 8. Thecrystal according to claim 2, which is characterized by the differentialscanning calorimetry chart in FIG.
 4. 9. A pharmaceutical compositioncomprising6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-onehydrochloride.
 10. The pharmaceutical composition according to claim 9,which is a Btk inhibitor.
 11. The pharmaceutical composition accordingto claim 10, which is an agent for the prevention and/or treatment of aBtk-related disease.
 12. The pharmaceutical composition according toclaim 11, wherein the Btk-related disease is an allergic disease, anautoimmune disease, an inflammatory disease, a thromboembolic disease, abone-related disease, or cancer.
 13. The pharmaceutical compositionaccording to claim 12, wherein the cancer is non-Hodgkin's lymphoma.